Summary

There is evidence in Xenopus and zebrafish embryos that the neural
crest/neural folds are specified at the border of the neural plate by a
precise threshold concentration of a Bmp gradient. In order to understand the
molecular mechanism by which a gradient of Bmp is able to specify the neural
crest, we analyzed how the expression of Bmp targets, the Msx genes, is
regulated and the role that Msx genes has in neural crest specification.

As Msx genes are directly downstream of Bmp, we analyzed Msx gene
expression after experimental modification in the level of Bmp activity by
grafting a bead soaked with noggin into Xenopus embryos, by
expressing in the ectoderm a dominant-negative Bmp4 or Bmp receptor in
Xenopus and zebrafish embryos, and also through Bmp pathway component
mutants in the zebrafish. All the results show that a reduction in the level
of Bmp activity leads to an increase in the expression of Msx genes in the
neural plate border. Interestingly, by reaching different levels of Bmp
activity in animal cap ectoderm, we show that a specific concentration of Bmp
induces msx1 expression to a level similar to that required to induce
neural crest. Our results indicate that an intermediate level of Bmp activity
specifies the expression of Msx genes in the neural fold region.

In addition, we have analyzed the role that msx1 plays on neural
crest specification. As msx1 has a role in dorsoventral pattering, we
have carried out conditional gain- and loss-of-function experiments using
different msx1 constructs fused to a glucocorticoid receptor element
to avoid an early effect of this factor. We show that msx1 expression
is able to induce all other early neural crest markers tested (snail,
slug, foxd3) at the time of neural crest specification. Furthermore, the
expression of a dominant negative of Msx genes leads to the inhibition of all
the neural crest markers analyzed. It has been previously shown that
snail is one of the earliest genes acting in the neural crest genetic
cascade. In order to study the hierarchical relationship between msx1
and snail/slug we performed several rescue experiments using dominant
negatives for these genes. The rescuing activity by snail and
slug on neural crest development of the msx1 dominant
negative, together with the inability of msx1 to rescue the dominant
negatives of slug and snail strongly argue that
msx1 is upstream of snail and slug in the genetic
cascade that specifies the neural crest in the ectoderm. We propose a model
where a gradient of Bmp activity specifies the expression of Msx genes in the
neural folds, and that this expression is essential for the early
specification of the neural crest.

Introduction

The neural crest originates at the border between the neural plate and the
future epidermis. It gives rise to numerous and diverse cell types, including
much of the peripheral nervous system, the craniofacial skeleton, and pigments
cells (for a review, see LaBonne and
Bronner-Fraser, 1999; Mayor
and Aybar, 2001). Although considerable progress has been made
recently in the molecular characterization of neural crest-inducing factors,
relatively little is known about the genetic cascade of transcription factors
that determine the specification of the neural crest at the neural plate
border.

The connection between the inductive molecules and the transcription
factors activated in the neural crest has not been elucidated. As described
above, one of the molecules that has a clear role in neural crest induction is
Bmp. To link the inductive molecules to the transcription factors, we have
started to analyse the role that downstream targets of Bmp could have on
neural crest specification. Most of the Bmp target genes identified to date
encode homeobox proteins, including msx1
(Suzuki et al., 1997),
msx2, vent1 (Gawantka et al.,
1995), vent2
(Onichtchouk et al., 1996) and
dlx5 (Miyama et al.,
1999). In this work, we have studied how Msx gene expression is
controlled in the neural folds and the role of msx1 in neural crest
development.

The expression of Msx genes is complex. In Xenopus embryos,
msx1 is initially expressed in ventral mesoderm and ectoderm, but
later becomes restricted to the neural folds and dorsal neural tube
(Suzuki et al., 1997). It has
been shown that msx1 can act as a ventralizing factor of the mesoderm
and as an inhibitor of nodal signaling
(Yamamoto et al., 2000;
Yamamoto et al., 2001).
Despite its expression in the neural fold, its function in neural crest
development has not been analyzed.

In order to understand how msx1 expression is regulated in the
prospective neural crest, we proceeded to inhibit Bmp activity and then
analyzed the expression of msx1. We inhibited Bmp activity by
grafting into Xenopus embryos beads soaked with noggin, by expressing
a dominant-negative form of Bmp or a dominant negative of its receptor in
Xenopus embryos, or by using several Bmp/Smad zebrafish mutants. Our
results show that inhibition of Bmp activity leads to an increase in
msx1 expression in Xenopus and zebrafish embryos. In
addition, by generating different levels of Bmp activity in animal cap
ectoderm, we show that there is a specific concentration of Bmp that can
induce msx1 expression. To study msx1 function in neural
crest specification and development, we carried out conditional gain- and
loss-of-function experiments using different msx1 constructs fused to
a glucocorticoid receptor element. Our results show that activation of
msx1 induces an expansion of the neural crest territory, as analyzed
by the expression of snail, slug and foxd3, whereas
expression of an msx1 dominant negative suppresses all neural crest
markers analyzed. By performing rescue experiments of the Msx genes
dominant-negative with inducible forms of snail and slug, we
show that msx1 lies upstream of the Snail family of genes in the
genetic cascade of neural crest specification. We propose a model whereby Msx
genes are induced by a gradient of Bmp activity and that this induction is
essential for neural crest specification.

Materials and methods

Xenopus and zebrafish embryonic manipulation

Embryos were obtained from adult Xenopus laevis by standard
hormone-induced egg laying and artificial fertilization
(Villanueva et al., 2002).
Embryos were staged according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1967)
and animal caps were carried out using eyebrow knives as indicated in Aybar et
al. (Aybar et al., 2003).
Zebrafish mutant embryos were obtained by intercrossing heterozygous parents;
alleles used were swrtc300,
sbndtc24 and
snhty68a.

Morpholino treatment in zebrafish

In order to inhibit mesoderm formation, the following combination of two
different spadetail (spt) and notail (ntl)
morpholinos (MO) was injected at the one-cell stage zebrafish embryo. The
spt MO was a kind gift of Sharon Amacher and Bruce Draper. The mix of
MO was a kind gift from Kate Lewis. The mix used had the final concentrations
of: ntl MO 1 mg/ml; spt MO#2 0.075 mg/ml; spt MO#1
0.75 mg/ml. The ntl MO sequence has been previously published
(Nasevicius and Ekker, 2000)
and the spt MO sequences are: spt MO 1,
5′-AGCCTGCATTATTTAGCCTTCTCTA-3′; and spt MO 2
5′-GATGTCCTCTAAAAGAAAATGTCAG-3′.

Plasmid constructs

Inducible DNA constructs of Msx genes were prepared by fusing the entire
coding regions of msx1 (amino acid residues 1-294,
Fig. 5A) to the ligand-binding
domain of the human glucocorticoid receptor (GR, amino acid residues 512-777).
A dominant-negative DNA construct (dnmsx) was prepared by fusing the
homeodomain region of msx1 (amino acid residues 156-294) to the GR
element (Fig. 5A). Coding
sequences were amplified by PCR with a high fidelity polymerase (Roche
Molecular Biochemicals, Mannheim, Germany) and the following primers:
msx1, 5′-ATGGGGGATTCGTTGTATGGATCGC-3′ and
5′-GAGCTCCGGACAGATGGTACATGCTGTATCC-3′; and
DNmsx1, 5′-GAATTCATGAGCCCACCCGCCTG-3′ and
5′-GAGCTCCGGACAGATGGTACATGCTGTATCC-3′.

msx1 fusion proteins and its phenotypic effects. (A) The
constructs used to produce the Msx genes fusion proteins are represented in
this figure. HD, msx homeodomain. GR, ligand binding domain of
glucocorticoid receptor. See Materials and methods for details. (B) Embryos
were injected with 700 pg of the indicated constructs, treated with
dexamethasone immediately after the injection and the phenotype was analyzed
at the tadpole stage. Anterior is towards the right. Top embryo, uninjected
control; middle embryo, embryos injected with msx-GR. Note the inhibition of
the anterior structures and ventralization of the embryo, similar to the
effect of injection Msx genes mRNA (not shown). Bottom embryo, embryo injected
with HDmsx-GR (dominant negative). Note that the effect, dorsalization, is
similar to the injection of dominant negatives of msx1 (not
shown).

The PCR products were purified and cloned into pGEM-T Easy vector
(Promega), EcoRI/SacI-digested and ligated with a
SacI/XhoI-digested GR fragment into a pCS2+ vector digested
with EcoRI/XhoI. Both fusion constructs were sequenced on
both strands at junction sites by automated DNA sequencing (BRC, Cornell
University, Ithaca, NY, USA). ΔBmpr and CM-Bmp4 constructs were kindly
donated by Dr K. W. Cho.

RNA microinjection, lineage tracing and dexamethasone induction

Dejellied Xenopus embryos were placed in 75% NAM containing 5%
Ficoll and one blastomere of two-cell stage embryos was injected with
differing amounts of capped mRNA containing 1-3 μg/μl lysine fixable
fluorescein dextran (40,000 Mr; FLDX, Molecular Probes) as
a lineage tracer. For the inhibition of Bmp activity, the dominant negatives
were injected in one animal blastomere of eight- to16-cell stage embryo. For
animal cap assays, mRNA was injected into the animal side of the two
blastomeres of two-cell stage embryos. Approximately 8-12 nl of diluted RNA
was injected into each embryo. Ethanol-dissolved dexamethasone (10 μM) was
added to the culture medium at stages 12 or 17, and maintained until the
embryos were fixed. To control the possible leakage of inducible chimeras, a
sibling batch of embryos were cultured without dexamethasone and processed for
in situ hybridization. One-cell stage swr/bmp2b mutant zebrafish
embryos derived from crosses of swr mutant homozygous adults
(Nguyen et al., 1998) were
microinjected with either chordin mRNA
(Miller-Bertoglio et al.,
1997) or a dominant negative Bmp type I receptor (ΔBmpr)
mRNA (Graff et al., 1994), as
previously described (Nguyen et al.,
2000).

Noggin treatment

Acrylic beads (Sigma) were incubated overnight with 100 μg/ml of noggin
protein (a kind gift from R. Harland). The beads where grafted into embryos at
the appropriate stage and the expression of several markers was later analyzed
by in situ hybridization. PBS-soaked beads were used as controls.

RNA isolation and RT-PCR analysis

Total RNA was isolated from embryonic tissue by the guanidine
thiocyanate/phenol/chloroform method
(Chomczynski and Sacchi, 1987),
and cDNAs were synthesized using AMV reverse transcriptase (Roche
Biochemicals) and oligo(dT) primer.

Primers for XSlug and H4 have previously been described
(Aybar et al., 2003). The
primers used to analyse Xenopus msx1 expression that amplify a 156 bp
product were 5′-GCTAAAAATGGCTGCTAA-3′ and
5′-AGGTGGGCTGTGTAAAGT-3′. PCR amplification with these primers was
performed over 28 cycles, and the PCR products were analysed on 1.5% agarose
gels. As a control, PCR was performed with RNA that had not been
reverse-transcribed to check for DNA contamination. Quantitation of PCR bands
was performed using ImageJ software (NIH, USA) on 8-bit greyscale JPG files
and the values were normalized to the H4 levels from the same sample and
expressed for comparison as relative intensities (sample/H4×10).

Results

Dynamic expression of msx1 within the neural crest

Although the expression pattern of msx1 has previously been
published in Xenopus and zebrafish embryos
(Suzuki et al., 1997;
Ekker et al., 1997), we
decided to analyse its expression pattern and compare it with Bmp4 expression.
Single and double in situ hybridization was performed in embryos at different
stages (Fig. 1). The expression
of Bmp4 has been already described in Xenopus embryos
(Schmidt et al., 1995), and a
dorsal (Fig. 1A) and lateral
view (Fig. 2B) are shown for
comparison with msx1 (msxe - Zebrafish Information Network)
expression at the mid gastrula stage (13). In Xenopus embryos, from
the early until the mid-gastrula stage (12), the expression of msx1
can be detected in the lateral and ventral ectoderm, as previously described
(Suzuki et al., 1997).
However, from the mid-gastrula stage onwards a progressive reduction in the
level of msx1 was observed in the ventral ectoderm, resulting in a
specific concentration of the label in the prospective neural crest
(Fig. 2D,E). At this stage (13)
a double in situ hybridization of msx1 and the neural crest marker
XSlug shows a clear overlap in their expression domains both in whole
mount (Fig. 2C,G), as well as
in sectioned embryos (Fig. 2F).
The msx1 expression domain includes, but is wider than, the neural
crest territory, probably being expressed in prospective placodes and the
neural plate border. A similar restriction from the ventral ectoderm to the
neural plate border in the expression of msxb was found
(Fig. 2H-K), confirming
previous publications (Ekker et al.,
1997; Cornell and Eisen,
2000). It should be mentioned that it is not clear whether
msxb is the orthologue of msx1
(Ekker et al., 1997).

msx1 expression is increased by inhibiting Bmp signaling in
Xenopus embryos. One blastomere of an eight- to 16-cell stage embryo
was injected with CM-Bmp4 mRNA (A,B) or ΔBmpr mRNA (D), or a bead soaked
with noggin was grafted near the neural fold of a stage 11 embryo (C), and the
expression of msx1 was analyzed at stage 17. Anterior is towards the
right; the injected side was recognized by FLDx staining and the operated side
by the bead, both are indicated by an arrowhead. (A) CM-Bmp4 mRNA (250 pg).
Note the stronger expression in the injected side. (B) CM-Bmp4 mRNA (500 pg).
Note the stronger and expanded expression in the injected side. (C) Embryo
grafted with a noggin soaked with 100 μg/ml of noggin (asterisk). Note the
expansion in expression at the grafted side. (D) ΔBmp4 mRNA (500 pg).
Note the stronger and expanded expression in the injected side. (E) Summary of
the results. The expression of msx1 was analyzed for each embryo
comparing the injected and uninjected side. Total number of embryos is 450.
Brackets indicate the domain of msx1 expression at the hindbrain
level.

Control of msx1 expression in the prospective neural crest
by Bmp signalling

It has been shown that the Msx genes are direct downstream targets of Bmp
signalling (Suzuki et al.,
1997; Maeda et al.,
1997; Yamamoto et al.,
2000; Ishimura et al.,
2000). We analysed msx1 expression after interfering with
Bmp activity. We inhibited Bmp activity in Xenopus embryos (1) by
injecting a dominant-negative form of Bmp4 (CM-Bmp4)
(Hawley et al., 1995) into one
blastomere of an eight- or 16-cell stage embryo, (2) by injecting a
dominant-negative form of the Bmp receptor (ΔBmpr) into one blastomere
of an eight- or 16-cell stage embryo, or (3) by grafting near the neural folds
of a stage 11 embryo a bead soaked with noggin. All these treatments produced
an expansion in the msx1 domain
(Fig. 2). It should be noted
that blocking Bmp signalling does not only expand the territory of
msx1 expression, but also increases its level of expression
(Fig. 2A,D). As the
noggin-soaked bead was grafted at stage 11 and the injections of the
dominant-negative mRNA were performed in the ectoderm, no effect on
gastrulation or mesodermal patterning was observed. An alternative way to
study the effect of decreasing Bmp activity on Msx gene expression is to use
different Bmp/Smad zebrafish mutants. The expression of msxb was
analysed in wild-type and mutant embryos. In zebrafish wild-type embryos at
the five-somites stage, msxb is expressed in two stripes lateral to
the dorsal axis (Fig. 3A),
similar to what has been described for Xenopus. In swr/bmp2
mutant embryos, the expression of msxb is expanded and moved to an
anterior domain (Fig. 3B) (see
also Schmid et al., 2000). In
sbn/smad5 and snh/bmp7 mutant embryos, msxb
expression is greatly and moderately expanded, respectively
(Fig. 3C,D). As in these mutant
embryos, not only the ectoderm but also the mesodermal patterning are
affected. A possible explanation of these results is that the expansion of the
neural plate/neural fold markers could be a secondary consequence of a primary
expansion of mesoderm, which in turn induces the neural markers in the
ectoderm. In order to test whether the expansion of the mesoderm played any
role in the expansion of the neural markers described here and in previous
publications (Nguyen et al.,
1998), we proceeded to inhibit the formation of dorsal mesoderm in
some of the mutant embryos and the neural markers were analysed. It has
previously described that krox20 (egr2b - Zebrafish
Information Network) is characteristically expanded as a ring in swr
mutant (Nguyen et al., 1998).
We analysed the expression of krox20, as a neural marker, and
myod as a mesodermal marker. The expression of both genes can be
clearly distinguished in wild type (Fig.
3E) and swr mutants
(Fig. 3F), showing the mutant a
characteristic expansion of both markers. In order to inhibit mesoderm
formation in the swr mutant, we proceeded to inject a mix of
ntl (Nasevicius and Ekker,
2000) and spt morpholinos. This injection lead to a total
inhibition in the expression of myod (100% of inhibition,
n=63), but no effect in the expansion of krox20 was observed
(Fig. 3G). These results
indicate that the expansion of the neural markers is not dependent on
mesodermal patterning.

Msxb expression is increased in Bmp/Smad mutant zebrafish embryos.
Wild-type and mutant zebrafish embryos were analyzed by whole-mount in situ
hybridization for the expression of Msxb at the five-somite stage. Lateral
views, anterior is upwards. (A) Wild-type embryos show the characteristic
dorsal expression in the embryo. (B) A swr mutant shows an expansion of the
Msxb territory in anterior regions. (C) A sbn mutant embryo shows a
dramatic ventral expansion in Msxb expression. (D) A snh mutant
embryo shows a moderate, lateral expansion in the expression of Msxb, where
the two domains of expression can be seen. (E) Flat-mount of a wild-type
embryo analyzed for the expression of Krox20 (arrows) and Myod (bracket). (F)
Swr mutant showing the ventral expansion of Krox20 (arrows) and Myod
(bracket). Note that the Myod expression is disorganized but can be found in
dorsal and ventral sides. (G) swr mutant injected with
ntl/sptl morpholinos. A complete absence of Myod expression
but an expansion in Krox20 (arrows) was observed. (H-J) swr mutant
embryos were injected with chordin mRNA and the expression of Msxb was
analyzed. Anterior is towards the left. (H) Control uninjected embryos show
the characteristic expansion of Msxb expression. (I) Embryos injected with 50
pg of chordin mRNA; note a reduction in Msxb expression. (J) Embryos injected
with 200 pg of chordin mRNA exhibit a total inhibition in the expression of
Msxb. Each experiment was repeated at least twice, with similar results.
Reducing Bmp signaling with ΔBmpr treatment yielded similar results to
those shown here for chordin.

The swr/bmp2 mutant exhibited an increase in the expression of
msxb (Fig. 3B) and a
reduction in the expression of neural crest markers
(Nguyen et al., 1998). In
order to test whether this msxb expression was dependent on remaining
Bmp activity in the swr/bmp2 mutant, we injected the embryos with
chordin or ΔBmpr mRNA and found similar results. After
injection of 50 pg of chordin mRNA, the expression of msxb
was significantly reduced, when compared with the uninjected control siblings
(Fig. 3H,I), and msxb
expression was not detectable after the injection of 200 pg of
chordin (Fig. 3J). The
expression of other neural markers, like krox20 or shh was
not affected in these injected embryos, ruling out a general effect on gene
expression. Taken together, our experiments using Xenopus and
zebrafish embryos strongly argue that Msx gene expression appears to be
dependent on a specific level of Bmp signalling.

So far, we have shown that there is no a direct correlation between the
level of Bmp activity and the expression of the Msx genes, as a reduction in
the first leads to an increase in the expression of the second. As we show in
Fig. 1, there are several
differences in the expression of Msx genes and bmp4. First, strong
expression of bmp4 is detected in the anterior neural plate border,
whereas almost no expression of msx1 is observed in that region
(Fig. 1, arrowhead). The
ventral epidermis shows a clear expression of bmp4
(Fig. 1B, arrow), and almost no
Msx gene expression (Fig. 1E,
arrowhead). The highest level of Msx gene expression can be observed in the
posterior neural folds (Fig.
1D,E, asterisk), while the level of Bmp4 expression in that region
is intermediate to the expression in the anterior neural plate border and the
epidermis (Fig. 1A,B,
asterisk). In conclusion, the levels of Bmp4 expression do not match exactly
those of msx1. However, the levels of bmp4 expression do not
necessarily correlate with Bmp activity levels. Furthermore, it is known that
Bmp binding molecules are secreted from the dorsal mesoderm, and in
consequence the levels of Bmp activity around the neural plate and neural
folds could be lower than those suggested by the levels of Bmp4 mRNA. Taken
together, these results prompted us to analyse directly the possibility that
msx1 transcription is induced at a certain threshold concentration of
a Bmp gradient.

It is known that neural crest can be induced in Xenopus by an
intermediate level of Bmp and Wnt signalling
(LaBonne and Bronner-Fraser,
1998; Marchant et al.,
1998; Saint-Jeannet et al.,
1997; Villanueva et al.,
2002). One-cell stage Xenopus embryos were injected with
a mixture of between 0 and 500 pg of dominant-negative Bmp4 (CM-Bmp4) mRNA and
50 pg of Wnt5a mRNA. Animal caps were dissected at stage 9, cultured until the
equivalent of stage 16 and the expression of Msx genes and the neural crest
marker XSlug were analysed by RT-PCR. As expected, XSlug
expression was induced at a specific concentration of CM-Bmp4 (100 pg,
Fig. 4A,B), lower or higher
amounts of CM-Bmp4 failed to induce strong XSlug expression,
confirming previous reports of induction of the neural crest by a gradient of
Bmp (Morgan and Sargent, 1997;
Marchant et al., 1998;
Nguyen et al., 1998;
LaBonne and Bronner-Fraser,
1998; Luo et al.,
2003). Interestingly, the highest level of msx1
expression was also induced at 100 pg of CM-Bmp4
(Fig. 4A,B). Low levels of
msx1 can be detected at other concentrations, which probably
represent the normal epidermal expression observed in untreated animal caps (0
ng of CM-Bmp4 mRNA, Fig. 4A,B).
These results strongly support the idea that, like the neural crest markers,
msx1 transcription is activated by an intermediate concentration in a
Bmp gradient.

Msx1 expression is specified by a threshold concentration of Bmp. One-cell
stage embryos were injected with a combination of 50 pg of Wnt5a mRNA and
different amounts of CM-Bmp4 mRNA, which are indicated in the figure. Animal
caps were dissected at stage 9 and the expression of msx-1, slug and
histone H4 was analyzed by RT-PCR when sibling embryos reached the neurula
stage 16. (A) Embryos and animal cap samples are shown. (B) Quantification of
the gel shown in A.

msx1 is required for neural crest specification

Given that msx1 is expressed in the prospective neural crest
territory and that it is induced by the same molecules that induce neural
crest, we investigated whether this gene might also function in neural crest
development. To overcome the early effects of msx1 in mesoderm
development, we used the inducible fusion constructs to the ligand-binding
domain of glucocorticoid receptor (GR) described previously
(Fig. 5A; see Material and
methods). To test if the function of msx1 was affected by the GR
domain in the fusion protein, we injected the msx-GR and the HDmsx-GR mRNA
into one-cell stage embryos, treated with dexamethasone immediately after the
injection (stage 5), and analyzed the phenotypes. No difference was detected
between the non-inducible and inducible msx1 constructs under these
conditions. msx-GR induced ventralization and anterior truncation
(Fig. 5B), and HDmsx-GR induced
dorsalization (Fig. 5B) as
previously published (Yamamoto et al.,
2000; Yamamoto et al.,
2001). However, when the induction was carried out at the gastrula
stage, none of these phenotypes was observed. This indicates that the early
function of msx1 is not altered in our fusion proteins, and that late
induction of them does not affect dorsoventral mesodermal patterning.
Furthermore, because the neural crest is specified around the late gastrula
stage, care was taken to induce the fusion constructs just before this stage
(stage 12) and analyse its effects at the early neurula stage (stage 18). This
is an important experimental approach as it has been shown that the neural
plate, but not the neural crest, is already specified at stage 12
(Servetnick and Grainger,
1991; Mancilla and Mayor,
1996; Woda et al.,
2003; Glavic et al.,
2004).

When mRNA encoding msx-GR was injected into one blastomere of a two-cell
stage embryo and then activated at stage 12, the expression of the neural
crest markers, slug, snail and foxd3, was augmented in more
than 70% of the injected embryos (Fig.
6A-C). Conversely, the activation at stage 12 of the inducible
dominant negative fusion, which contains the Msx gene homeodomain (HDmsx-GR),
impaired the expression of slug, snail and foxd3 in more
than 75% of the injected embryos (Fig.
6F-H). These results indicate that msx1 is required for
the expression of the neural crest markers. We then analyzed whether the
expansion of the neural crest territory was made at the expense of the neural
plate or the epidermis. The expression of the neural plate marker
Sox2 and the epidermal marker XK81a was analyzed after the
activation of msx-GR at stage 12. The results show that there was an
inhibition in the expression of Sox2 in about 60%
(Fig. 6D) and of XK81a
in 63% (Fig. 6E) of the
injected embryos. This result indicates that msx1 overexpression can
transform the neural plate and epidermal cells that surround the prospective
neural crest territory into neural crest cells. We should mention that
although many injections were localized at the center of the neural plate or
epidermis, we never observed ectopic expression of neural crest markers in
those territories. These results suggest that msx1 cannot transform
ectodermal cells by itself into neural crest cells, but probably requires
additional co-factors that are present near the neural crest region. In
support of this observation we never induced the expression of neural crest
markers in animal caps injected with msx1 mRNA, as analyzed by RT-PCR
(three independent experiments).

msx1 participates in the early specification of the neural crest.
One blastomere of a two-cell stage embryo was injected with 700 pg of msx-GR
mRNA (A-E), with 700 pg of HDmsx-GR mRNA (F-J) or with different combinations
of both mRNAs (K-P), treated with dexamethasone at stage 12.5. Embryos were
fixed at stage 18/19 and the expression of several genes was analyzed. The
arrowheads indicate the injected side that contained FLDx (see Materials and
methods). Anterior is towards the right. (A-C,F-H) Neural crest markers. (A-C)
Notice the expansion of the markers on the side injected with msx-GR. (A)
XSlug expression (n=44, 68% of expansion). (B)
XSnail expression (n=60; 80% of expansion). (C)
foxd3 expression. (n=52, 61% of expansion). (F-H) Note the
inhibition in the expression of the neural crest markers injected with
HDmsx-GR. (F) XSlug expression (n=57, 65% of inhibition).
(G) XSnail expression (n=42, 69% of inhibition). (H)
foxd3 expression (n=66, 64% of inhibition). (D)
XSox-2 expression in embryos injected with Msx-GR. Note the
inhibition in the expression (n=63, 38% of inhibition). (I)
XSox2 expression in embryo injected with HDmsx-GR. Note the expansion
in the expression the injected side (n=54, 39% of expansion). (E)
XK81a expression in embryos injected with Msx-GR. Note the inhibition
in the expression (n=57, 28% of inhibition). (J) XK81a
expression in embryos injected with HDmsx-GR. Note the expansion in the
expression (n=62, 32% of expansion). (K-M) Embryos were injected with
500 pg of HDmsx-GR mRNA and 250 pg of msx-GR (ratio 2:1). Note the partial
rescue in the expression of the neural crest markers. (N-P) Embryos were
injected with 500 pg of HDmsx-GR mRNA and 500 pg of msx-GR (ratio 1:1). Note
the rescue in the expression of the neural crest markers. (G) Summary of the
expression of XSlug. The injected and uninjected side was analyzed
for each embryo. Number of embryos analyzed for XSlug expression:
215. Note that the strong rescue (73%) was reached with a proportion of 1:1
for the injected mRNAs. Similar values of rescue were obtained for the other
neural crest markers (69% for foxd3, total number is 220; 67% for
XSnail, total number is 225).

We then inhibited msx1 function and examined the effect on the
neural plate and epidermis. Embryos were injected with HDmsx-GR mRNA into one
blastomere of a two-cell stage embryo, induced at stage 12 and the expression
of Sox2 and Xk81a was analyzed. We observed an expansion of
Sox2 (Fig. 6I) and
XK81a (Fig. 6J)
expression on the injected side of 58 and 71% of the embryos, respectively.
Taken together, these results show that the function of msx1 at the
late gastrula stage is to specify the fate of the ectodermal cells as neural
crest cells, and when its function is inhibited the prospective neural crest
cells are transformed into neural plate and epidermis. This function of
msx1 is coherent with its expression in the prospective neural crest
cells.

To show that the msx1 dominant-negative (XHDmsx1-GR) specifically
inhibits msx1 function, we performed rescue experiments. Embryos were
injected in one blastomere at the two-cell stage. The inducible constructs
were activated at stage 12, and the expression of the neural crest markers,
XSlug, foxd3 and XSnail was analysed at stage 18. The
injection of XHDmsx1-GR inhibited in greater than 65% of the embryos the
neural crest marker expression (Fig.
6Q, for simplicity only the percentage of XSlug
expression are indicated in the graphic, but similar percentages were obtained
for foxd3 and XSnail expression). However, the co-injection
of XHDmsx1-GR and msx-GR was able to rescue the expression of the neural crest
markers in a dose-dependent manner. When XHDmsx1-GR and msx-GR were injected
in a proportion of 2:1, the inhibition in the expression of the neural crest
markers was reduced to 50% (Fig.
6K-M,Q), but when equal amounts of both constructs were
co-injected, the inhibition of the neural crest markers was rescued in more
than 80% (Fig. 6N-P,Q). As
expected, the injection of msx-GR alone produced an expansion in the
expression of the neural crest markers in more than 70% of the embryos
(Fig. 6Q). Thus, we conclude
that the phenotypic effects of the inducible msx1 dominant-negative
reflect a modulation of the natural msx1 target genes.

A combination of HDmsx-GR and XSlug-GR was injected into one blastomere of
two-cell stage embryos, induced at stage 12 and the expression of XSlug,
foxd3 and XSnail was analysed. Different proportions of mRNA of
the two constructs were injected. As expected the injection of HDmsx-GR alone
produced a strong inhibition in the expression of the neural crest markers
(Fig. 7G); however,
co-injection of XSlug-GR rescued this effect in a dose-dependent manner
(Fig. 7A-G). The injection of
XSlug-GR leads to an expansion of the neural crest territory
(Fig. 7G), as previously
reported (Aybar et al., 2003).
Note that when equal amounts of HDmsx-GR and XSlug-GR are used, XSlug
expression is rescued in about 70% of the embryos
(Fig. 7A,G). Similar results
were obtained for other neural crest markers (foxd3,
Fig. 7B, 72% rescued;
XSnail, Fig. 7C, 68%
rescued). However, in the opposite experiment, co-injection of a dominant
negative slug (XSlugZnF-GR) (Aybar
et al., 2003) with msx-GR, no significant rescue in the expression
of the neural crest markers was observed (XSlug expression was
rescued in less than 10% of the embryos, n=67). We conclude that
msx1 function lies upstream of XSlug function in the genetic
cascade of neural crest specification.

msx1 lies upstream of XSlug and XSnail in the
cascade leading to neural crest development. Embryos were injected in one
blastomere at the two-cell stage with different combinations of HDmsx-GR and
XSlug-GR (A-G) or XSnail-GR (H-N), induced at stage 12 and the expression of
the neural crest markers XSlug (A,D,H,K), foxd3 (B,E,I,L)
and XSnail (C,F,J,M) was analyzed at stage 18. Anterior is towards
the right. The injected side detected by fluorescein staining is indicated by
an arrowhead. (A-C) Embryos were injected with 500 pg of HDmsx-GR mRNA and 500
pg of XSlug-GR (ratio 1:1). Note the rescue in the expression of the neural
crest markers. (D-F) Embryos were injected with 250 pg of HDmsx-GR mRNA and
750 pg of XSlug-GR (ratio 1:3). Note the rescue in the expression of the
neural crest markers. (G) Summary of the expression of XSlug. The
injected and uninjected side was analyzed for each embryo. Number of embryos
analyzed for XSlug expression is 225. Note that the strong rescue
(65%) was reached with a ratio of 1:1 for the injected mRNAs. Similar values
of rescue were obtained for the other neural crest markers (72% for
foxd3, total number is 225; 68% for XSnail, total number is
235). (H,I) Embryos were injected with 500 pg of HDmsx-GR mRNA and 500 pg of
XSnail-GR (proportion of 1:1). Note the rescue in the expression of the neural
crest markers. (K-M) Embryos were injected with 250 pg of HDmsx-GR mRNA and
750 pg of XSnail-GR (proportion of 1:3). Note the rescue in the expression of
the neural crest markers. (N) Summary of the expression of XSlug. The
injected and uninjected side was analyzed for each embryo. Number of embryos
analyzed for XSlug expression is 215. Note that the strong rescue
(67%) was reached with a ratio of 1:1 for the injected mRNAs. Similar values
of rescue were obtained for the other neural crest markers (51% for
foxd3, total number is 202; 62% for XSnail, total number is
215).

We performed similar rescue experiments with XSnail. Embryos were
co-injected with HDmsx-GR and XSnail-GR and a dose-dependent rescue of the
effect on neural crest markers was observed
(Fig. 7H-N). Once more, the
optimal rescue was obtained when equal amounts of both mRNA were used,
reaching a nearly 70% rescue in the expression of XSlug, foxd3 and
XSnail. Again, compared with this level of rescue, we failed to
produce a significant rescue by the co-injection of a dominant-negative
XSnail (XSnailZnF-GR) (Aybar et
al., 2003) and msx-GR (less than 10% rescued for the expression of
XSlug, n=104). These results support the idea that msx1 is
upstream of XSnail in the cascade of specification of the neural
crest. Thus, as XSnail is one of the earliest genes to be expressed
and to work in the neural crest genetic cascade, we propose that msx1
could be the earliest gene to be activated in this cascade.

We present evidence that supports the idea that a specific level of Bmp
activity leads to Msx gene transcription. In addition, we show that this level
of Bmp activity corresponds exactly with the level that is able to induce
neural crest cells. Thus, Msx gene transcription is dramatically increased in
the ectoderm by a level of Bmp activity intermediate to the level required to
induce neural plate or epidermis. This high level of Msx genes, induced at a
precise and intermediate threshold of Bmp signaling, is required to induce
neural crest cells.

There are many reports that support the idea that a gradient of Bmp
activity divides the ectoderm into neural plate, neural crest and epidermis.
The molecular mechanism by which different levels of Bmp are able to activate
the transcription of different genes and in turn specify different tissues is
unknown. One possibility is that Bmp activates all its downstream target genes
in a linear way; thus, the gradient of Bmp should be transformed into a
gradient of all of its targets genes, including Msx genes, with high and low
levels in the ventral and dorsal sides, respectively. Then this gradient of
Msx genes should specify the neural crest at an intermediate level by an
unknown mechanism. In this work, we show that this is not the case for Msx
genes, as this gene is induced by a precise and intermediate level of Bmp
activity. Thus, we rule out the alternative that the neural crest is specified
by an intermediate concentration of Msx protein within a gradient. Instead, we
support the idea that the gradient of Bmp is immediately transformed into the
activation of Msx genes at a precise threshold.

The molecular mechanism by which Msx genes are expressed only at
intermediate levels of Bmp activity is unknown. One possibility is the
presence of Bmp receptors with different affinities for its ligands and with
different downstream targets, being Msx genes a specific target for a specific
receptor. Another alternative is the activation at high levels of Bmp activity
of a repressor of Msx gene transcription. This alternative is supported by the
observation of an early and transient expression of msx1 and
msxb in the ventral ectoderm, which could be inhibited at later
stages by this hypothetical repressor. Additional experiments are required to
distinguish between these and other alternatives.

Role of Msx genes in neural crest specification

Our gain- and loss-of-function experiments show that Msx genes are required
for the early specification of the neural crest. Inhibition of Msx gene
function at the time of neural crest specification by use of an inducible
dominant negative, leads to inhibition in the expression of the earliest
neural crest markers known like snail, slug and foxd3.
Activation of Msx genes just prior to neural crest specification leads to an
expansion of the endogenous neural crest territory; however, we never observed
isolated regions of neural crest marker induction within the neural plate or
epidermis. This result suggests that Msx genes work together with other
factors, present in the neural plate border, to specify neural crest cells.
This explanation is also supported by our inability to induce neural crest
markers in animal caps injected with Msx genes. Taken together, we propose
that the Bmp gradient induces at the neural plate border the expression of Msx
genes and another factor, and that both are required to activate the genetic
cascade of neural crest specification. This additional factor could also be
activated by the Wnts, Fgf or retinoic acid signaling, as it is known that
they are required for neural crest induction
(LaBonne and Bronner-Fraser,
1998; Villanueva et al.,
2002; Saint-Jeannet et al.,
1997; Luo et al.,
2001a; Luo et al.,
2001b). One possible candidate for this additional factor could be
pax3, as it is known that it is expressed at the neural plate border
in a domain slightly broader than the neural crest territory, and it is able
to activate the expression of neural crest markers
(Bang et al., 1999;
Mayor et al., 2000). In our
animal cap gradient experiment, Wnt signaling was required to induce neural
crest and msx1 expression (Fig.
4E), as widely reported
(LaBonne and Bronner-Fraser,
1998; Villanueva et al.,
2002; Saint-Jeannet et al.,
1997; Luo et al.,
2001a; Luo et al.,
2001b; Honoré et al.,
2003). In addition, it has been observed that there is a
synergistic effect between Bmp and Wnt signaling in the induction of Msx genes
in culture cells (Willert et al.,
2002). The ability of Msx genes to induce neural crest is time
dependent. When the activation of Msx genes is performed before gastrulation,
it does not promote neural crest development, but instead promotes epidermal
development (Suzuki et al.,
1997); when Msx genes are activated after gastrulation (stage 17),
once the neural crest is specified
(Mancilla and Mayor, 1996;
Aybar and Mayor, 2002), no
increase in neural crest markers is observed (not shown).

Once the neural crest genetic cascade is activated by Msx genes, the
expression of specific genes that are able to confer neural crest identity is
induced. One of the earliest genes in this cascade seems to be snail
(Aybar et al., 2003), as it is
the only gene identified so far whose expression in animal caps is able to
specifically induce the expression of early and late neural crest markers. The
expression of genes such as Meis, Pbx, foxd3 and Zic family members,
not only trigger the expression of neural crest markers, but also induce the
expression of neural plate markers (Sasai
et al., 2001; Nakata et al.,
2000; Mizuseki et al.,
1998; Nagai et al.,
1997; Nakata et al.,
1997; Nakata et al.,
1998; Maeda et al.,
2002). In addition, snail seems to lie upstream of
slug in the neural crest genetic cascade
(Aybar et al., 2003). In this
work, we rescued the effect of an Msx gene dominant negative by snail
or slug co-injection, but we were not able to rescue the effect of a
slug or snail dominant negative by msx1
co-expression. Taken together, these results strongly support the conclusion
that Msx genes are upstream of snail/slug in the specification of the
neural crest cells. This conclusion is consistent with the fact that Msx genes
are a direct target of Bmp, which is one inducer of the neural crest.

It should be noted that the expression of Msx genes includes the
prospective neural crest territory, but also encompasses cells adjacent to the
neural crest. Those adjacent cells could be placodal and dorsal neural tube
cells. Thus, although we have demonstrated a clear role for Msx genes in early
neural crest specification, other experiments remain to be performed to
investigate the role of Msx genes in other neural fold cells types.
Interestingly, there is evidence that indicates that the preplacodal field,
which is adjacent to the neural crest, is also specified by a precise
threshold concentration of the Bmp gradient (A. Glavic and R.M.,
unpublished)

Loss of function of Msx genes in the mouse, by knocking out the gene or use
of antisense oligonucleotides, produced a wide range of phenotypes, many of
them related to the development of neural crest derivatives
(Foerst-Potts and Sadler,
1997; Jumlongras et al.,
2001; Satokata and Maas,
1994). However, analysis of these results is complicated by the
presence of three Msx genes, which could operate in a redundant manner. In
addition to the early role of Msx genes in neural crest specification, these
genes play a role in the control of apoptosis
(Gomes and Kessler, 2001;
Marazzi et al., 1997). The
more complex pattern of expression within the neural folds observed at neurula
stages is probably related to its apoptotic function in the neural crest
(C.T., M.J.A. and R.M., unpublished).

In conclusion, the dynamic expression of Msx genes during embryonic
development is probably a consequence of a complex system of transcriptional
regulation and reflects the multiple functions that this gene plays in several
developmental processes. We have unravelled one of its roles in early neural
crest development and have shown how its expression is controlled in these
cells.

Acknowledgments

R.M. thanks J. Eisen and all members of her laboratory for the zebrafish
work performed at the University of Oregon. Special thanks to S. Cheesman and
C. Miller for his generous help. We thank N. Ueno for the wild type
msx1 clone; and Y. Sasai, R. Grainger, K. Cho, S. Amacher, B, Draper,
K. Lewis, J. Muyskens and T. Sargent for reagents used in this research. This
investigation was supported by an International Research Scholar Award from
the Howard Hughes Medical Institute to R.M., and by grants from Fondecyt
(#1020688), the Millennium Program (P99-137F) and the MRC. C.T. was supported
by a PhD fellowship from the Millennium Program and MJA by grants from
Fondecyt (#3010061) and Fundación Antorchas (14169-3). M.C.M. and
V.H.N. were supported by NIH grant GM56326.

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